Thyroid follicular cells perform multiple functions that depend upon epithelial polarity, including the secretion of thyroglobulin (Tg) into the lumen of thyroid follicles, uptake of iodide and its use for covalent modification of Tg, and the manufacture and delivery of thyroid hormones to the bloodstream. Defects in several of these steps are well-recognized causes of hypothyroidism. This application focuses on dissecting these processes at the cellular level. A major area of concentration is the traffic of newly synthesized Tg from its site of synthesis in the endoplasmic reticulum (ER) to its delivery in the follicle lumen. New evidence suggests that initial folding of nascent Tg-is a complicated process involving a family of helper proteins known as molecular chaperones in the ER. A second area is the protein targeting mechanism that allows Tg to be directed to the follicle lumen instead of the bloodstream. Using cultured thyroid epithelial monolayers on porous filters, which allow experimental access to both apical and basolateral surfaces, the investigators have now identified (by purifying and partial sequencing) a second protein that --unlike Tg-is secreted with basolateral preference. Thus, thyrocytes exhibit true intracellular sorting in the distal secretory pathway. The investigator hypothesizes that sorting signals in regulated secretory proteins lead to apical-specific targeting in thyrocytes; specific transfection experiments are proposed to test this idea. A third area is the distribution and delivery of apical and basolateral plasma membrane proteins that are essential for hormonogenesis; a straightforward experimental plan is proposed to examine thyroid cell surface polarity. Finally, a recent breakthrough indicates that the filter-polarized thyrocytes both synthesize T4 and secrete it with a marked basolateral preference. The investigators hypothesize that the polarized secretion of thyroxine is mediated by specific transporters in the thyrocyte basolateral membrane.In the last Specific Aim the investigator proposes an initial characterization of this transport using a variety of pharmacologic agents that act as thyroid inhibitors and stimulators.